Analyses of the levels of proteins in mammalian serum are widely used as indications of various diseases and abnormal physiological conditions. A subnormal level of albumin, for example, is an indication of renal disease, while high albumin is a characteristic of dehydration. Similarly, an elevated level of pre-.beta. lipoprotein can be an indication of chronic alcoholism or of hyperestrogenism, and elevated levels of .beta.-lipoprotein can be indicative of high cholesterol.
Proteins are readily separated by electrophoresis, and both slab gel electrophoresis and capillary electrophoresis have been used. The advantage of capillary electrophoresis is that very small samples can be analyzed, and because of the high wall surface-to-volume ratio in a capillary the heat generated by the electric current is rapidly dissipated, which permits separations to be peformed at high voltages and therefore shorter separation times. Open capillary zone electrophoresis, in which the separation medium is a buffer solution, is particularly useful since the capillary can be easily conditioned and filled with the solution for each use.
The electrokinetic potential at the capillary wall surface is a prominent factor in the movement and separation of the proteins upon imposition of the electric field. This movement and separation can be changed by the interactions between the proteins and the capillary wall. Separations run at high or low pH, which place a net charge on the proteins, may minimize the interaction between the proteins and the capillary wall. These interactions are particularly strong in capillaries made of fused silica, causing adsorption of the proteins at the capillary wall. The adsorption has an influence on the electroosmotic flow by changing the charge density of the wall surface. Changes in the electroosmotic flow in turn cause changes in migration times, peak resolution and reproducibility. These considerations also apply, although often to a lesser extent, in slab electrophoresis, particularly with the use of thin slabs.
Efforts to improve the separation by suppressing the interaction between the proteins and the capillary wall or other wall enclosing the separation zone have resulted in the development of a variety of treatments, additives and operating conditions. One technique is to use a buffer having a pH that is higher than the isoelectric points of the sample proteins, thereby imparting a net negative charge to the proteins to repel them from the negatively charged wall. A disadvantage of this method is the risk of hydrolysis and other structural changes in the proteins. Strongly basic buffers with high salt concentrations have also been used as a means of blocking ionic interactions between the proteins and the wall. High salt concentrations however increase the conductivity of the buffer and hence the rate of heat generation inside the capillary. A further alternative has been to coat the wall to mask or neutralize the potential, and coatings for this purpose have included polyacrylamide, glycol, and pentafluorobenzoyl groups. Disadvantages of coated walls include low reproducibility due to the gradual deterioration of the coating over repeated use, and a reduction in the speed of the analysis due to the elimination of the electroendosmotic flow component. A still further alternative has been the inclusion of borate ion in the running buffer to form complexes with the the sugar moieties of glycoproteins and thereby neutralize their charge. The use of borate ion entails a risk of denaturing certain proteins, particularly immunoglobulins, and thereby creating a separate peak. Also, borate ion increases the conductivity of the buffer, limiting the electric field and the speed with which the separation can be achieved.